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Biology of Reproduction 61, 1267-1274 (1999)
© 1999 Society for the Study of Reproduction, Inc.

Articles

Cyclic Modulation of Integrin Expression in Bovine Endometrium1

Sarah Kimminsa, and Leslie A. MacLaren2,a

a Animal Science Department, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada B2N 5B1


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are heterodimeric glycoproteins involved in cell-cell and cell-extracellular matrix adhesion. In this study, the spatial and temporal distribution of selected integrins and extracellular matrix proteins was determined in bovine endometrium from cycling and ovariectomized animals using indirect immunohistochemistry. The expression of integrins {alpha}6 and {alpha}vß3 was estrous cycle-dependent. Strong immunostaining for {alpha}vß3 occurred in the basement membrane region of intercaruncular luminal epithelium except on Day 16 (P < 0.05). Staining of subepithelial stromal cells declined in diestrous samples (P < 0.05). In all samples, there was reduced {alpha}vß3 reactivity in the caruncles. Staining for {alpha}6 decreased in the epithelial basement membrane at proestrus through estrus (Days 18–0). Expression of integrin subunits {alpha}3 and {alpha}4 was cycle-independent. Moderate staining for {alpha}3 was detected on epithelium and {alpha}4 was present on stromal cells. The distribution of ß1 suggested dimerization with {alpha}3, {alpha}4, and {alpha}6. Laminin was detected in the epithelial and vasculature basement membranes. Collagen IV was present in the glandular epithelium basement membrane and subepithelial stromal cells, whereas fibronectin was found only in the stroma. Estrous cycle-dependent distribution and expression of {alpha}vß3 and {alpha}6 suggest their regulation by ovarian steroids, growth factors, and prostaglandins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ruminant endometrium is a complex tissue composed of distinct cell types representing many lineages. It is generally categorized as caruncular, referring to the thickened subepithelial burrs where hemotropic placentation proceeds, or intercaruncular, referring to the glandular regions between caruncles [1]. Under the influence of ovarian steroids, the endometrium undergoes cyclic changes that ultimately lead to nourishment and attachment of the embryo following fertilization [2]. Through the estrous cycle, we expect growth and regression of the uterus, and changes in local hormone and growth factor expression [3–5]. It would be expected that, as in other species [6], there would also be cyclic changes in cell-cell and cell-substratum interactions to facilitate the transitions from one stage of the estrous cycle to the next, and from diestrus to pregnancy.

The social interactions of cells with each other and with proteins in the extracellular matrix (ECM) are partly mediated by a large family of adhesion proteins, named integrins [7]. Integrins are transmembrane glycoproteins composed of {alpha} and ß subunits that exist in close association with the cytoskeleton and signaling proteins [7–9]. Since the initial characterization of integrins in the 1980s, 22 integrin receptors have been identified, and these comprise at least 16 types of {alpha} subunits and 8 types of ß subunits [10]. Cell processes affected by the receptor are determined by the {alpha}ß combination, and not all {alpha} subunits can dimerize with every ß subunit. Each integrin receptor has specific effects on tissue architecture and cell processes, such as migration, proliferation, and signal transduction [10, 11]. Integrin ligands, a structurally diverse group of ECM proteins that include fibronectin, laminin, collagen, vitronectin, and osteopontin, can affect the spatial and temporal distribution of integrins [12, 13]. In turn, steroids, growth factors, and pharmacological agents modulate the expression and actions of these receptors [14–16].

In mice, pigs, and humans, integrins undergo estrous cycle-dependent modulation, indicating that these proteins may be good markers of uterine receptivity [17–19]. A convenient definition of receptivity is that period during which embryo survival and attachment is favored: In the cow this includes the first 16 days of the estrous cycle, and, if the embryo has successfully signalled its presence to the dam, Days 17–19, with attachment beginning about Day 19 [20]. If pregnancy is not recognized, the endometrium undergoes the changes associated with the follicular phase of the estrous cycle under the influence of estrogen. The expression and distribution of integrins in bovine endometrium during the receptive and nonreceptive stages of the estrous cycle have not been studied.

In the present study, immunohistochemical techniques were used to characterize integrin expression in bovine endometrium throughout the estrous cycle. Antibodies directed against the ECM proteins laminin, fibronectin, and collagen type IV were used to examine the distribution of these integrin ligands in relation to the distribution of integrin receptors. In addition, ovariectomized animals treated with either estradiol-17ß (E2), progesterone (P4), no treatment, or E2 and P4, were examined to determine the effects of the ovarian steroids on integrin expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

All procedures performed were in accordance with the guidelines of the Canadian Council on Animal Care and were reviewed and approved by the Nova Scotia Agricultural College Animal Care and Use Committee. Sexually mature, mixed-beef-breed animals (n = 39) were slaughtered at government-inspected abattoirs on Days 0 (estrus), 1, 3, 6, 10, 14, 15, 16, 17, 18, 19, and 20 of the estrous cycle, three animals each day except Days 14 (n = 4) and 16 (n = 5). To confirm the stage of the estrous cycle, animals were observed for estrous behavior, and blood samples taken at estrus and slaughter were assayed for P4 by RIA using a Coat-A-Count kit (Diagnostic Products Corporation, Los Angeles, CA). Also, ovarian dating was performed at collection according to the criteria of Ireland [21]. After palpation to confirm cyclicity, an additional 10 animals were ovariectomized (OVX). Bilateral ovariectomies were performed via an incision through the left para lumbar fossa after local anesthesia with lidocaine 2% (Vetoquinal Canada Inc., Joliette, PQ, Canada) in an inverted L block. One week after surgery, animals received one of the following treatments for 12 days: E2 (OVX-E; 24-mg implant, Compudose; Eli Lilly, Quebec City, QC, Canada, n = 3), P4 (OVX-P; 1.9-g P4-releasing intravaginal device [PRID]; Sanofi, Cambridge, ON, Canada, n = 2), both E2 and P4 (n = 2, OVX-E+P), or the control, not treated (OVX-no T, n = 3). PRIDs were changed after 6 days to maintain circulating P4 levels greater than 4 ng/ml and were removed at slaughter. Uteri from all animals were dissected into 1-cm3 cross-sectioned blocks, frozen in liquid nitrogen (-179°C) at the collection site, and transferred to an ultra-low-temperature freezer (-80°C) for storage.

Antibodies

Monoclonal antibodies specific to integrins {alpha}4, {alpha}6, and {alpha}vß3, and a polyclonal antibody specific to {alpha}3 were purchased from Chemicon (Temecula, CA). A ß1 monoclonal antibody developed by Dr. C. Damsky was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). ECM proteins fibronectin (Chemicon) and collagen type IV and laminin (ICN Biomedical Inc., Aurora, ON, Canada) were localized using polyclonal antibodies. The Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) was used for signal amplification of integrins {alpha}4 and {alpha}vß3. Horseradish peroxidase (HRP)-conjugated goat-anti-rabbit IgG (Cedarlane Laboratories Ltd., Hornby, ON, Canada) was used for immunolocalization of polyclonal antibodies, and HRP-conjugated goat-anti-rat IgG (Cedarlane) was used to localize anti-integrin {alpha}6. For co-localization of integrin {alpha}6 and laminin, fluorochrome-conjugated anti-rabbit IgG (Alexa 594) and anti-rat IgG (Alexa 488) probes were obtained from Molecular Probes Inc. (Eugene, OR). Controls included normal rabbit serum (ICN) and purified mouse and rat IgG (Chemicon). Optimal dilutions were used to maximize immunostaining (Table 1).


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  		TABLE 1. Primary antibodies

Immunohistochemistry

Immunoperoxidase staining for integrins was performed on 5- to 8-µm cryostat cross sections of endometrium. Serial sections were mounted on 3-aminopropyl-triethoxysilane-coated-Superfrost Plus slides (Fisher Scientific, Whitby, ON, Canada) and then fixed in acetone for 10 min. Cut sections were stored at -80°C until use. Sections were blocked with 2% BSA in PBS (pH 7.2 to 7.4) for 45 min. After a PBS rinse, primary antibody was allowed to bind for 2 h; then the slides were washed in PBS 3 times for 3 min each, and secondary antibody was applied and allowed to react for 45 min. Immunostaining was visualized using the chromogen metal enhanced diaminobenzidine (Pierce, Rockford, IL). Slides were then counterstained with Mayer's hematoxylin (Electron Microscopy Sciences, Fort Washington, PA) and coated with Aquaperm mounting medium (Fisher). Slides were viewed on an Olympus BH-2 microscope equipped with an Olympus omPC camera (Olympus Optical Company, Tokyo, Japan) and photographed on Ektachrome 64T film (Eastman Kodak, Rochester, NY), or images were captured using a Pixera digital camera (Pixera Corporation, Los Gatos, CA). In double-fluorescence experiments, the primary and fluorochrome-conjugated secondary antibodies were applied sequentially to the same tissue section. After being washed in PBS, the sections were mounted in Vectashield Mounting Medium (Vector Laboratories), and images were captured by a confocal laser scanning scope (Carl Zeiss, Oberkochen, Germany). All experiments were replicated three times for each animal on different days.

Statistical Analysis

When cycle-dependent integrin expression was perceived, sections were scored by two independent observers on a six-point scale (1, negative; 2, low; 3, moderate; 4, intermediate; 5, high; and 6, very high) for staining intensity. Statistical comparisons between cycle days were made by the Kruskal-Wallis test of population medians [22]. When significant effects were observed, Mann-Whitney's two sample rank test [22] was used to test the equality of population medians.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cycle-dependent Integrin Expression

There was strong expression of integrin {alpha}vß3 in the basement membrane regions of intercaruncular luminal epithelium during metestrus, proestrus, and estrus (Fig. 1). Shallow glands stained moderately in the basement membrane region at all stages of the cycle (Fig. 1, B and D). The number of subepithelial stromal cells expressing {alpha}vß3 declined (P < 0.05) during diestrus (Fig. 2A). On Day 16, {alpha}vß3 was not detected in the basement membrane region of luminal epithelium (P < 0.05; Figs. 1C and 2B), and pericellular staining of subepithelial stromal cells was weak or absent (score 1–2; Fig. 1C). For all cycle days examined, expression of {alpha}vß3 was low or very weak in caruncular regions, with no distinct staining in the basement membrane region of luminal epithelium or on subepithelial stromal cells (Fig. 1, B–D). Moderate staining of {alpha}vß3 in blood vessels and myometrium was present for all cycle days examined. Steroid-treated ovariectomized animals had reduced levels of {alpha}vß3 expression in comparison to control OVX animals receiving no treatment (Figs. 1E and 2C).



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  		FIG. 1. Immunohistochemical localization of cycle-dependent integrin {alpha}vß3 in cryosections of bovine endometrium. Immunoreactivity (indicated by brown color) was localized to the basement membrane region (closed arrows) of luminal epithelium (LE) of intercaruncular regions (ICAR). Diffuse staining was detected throughout the stroma (S), and staining was reduced in, or absent from, the basement membrane region (open arrows) of caruncles (CAR). GE, Glandular epithelium. A) Day 3, B) Day 6, C) Day 16, D) Day 17, E) OVX-E+P, F) negative control treated with mouse IgG. Nuclei counterstained with hematoxylin. The bars represent 50 µm



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  		FIG. 2. Altered expression of the integrin {alpha}vß3 in cycling and OVX animals. A) Estrous cycle effect on {alpha}vß3 expression in the subepithelial (SE) stroma. B) Estrous cycle effect on {alpha}vß3 expression in the basement membrane (BM) region. C) Sex steroid effects on {alpha}vß3 expression in ovariectomized animals. Bars not sharing the same letter are significantly different (P < 0.05)

Antibodies to {alpha}6 stained uterine epithelium and a subpopulation of stromal cells, primarily endothelial cells, in both caruncular and intercaruncular regions (Fig. 3). Immunoreactivity for {alpha}6 was concentrated in the basement membrane. Stromal cells and glands located within the compact stroma stained intensely for {alpha}6, whereas staining was moderate in the loosely packed stroma. Through metestrus and diestrus (Days 1–16; P < 0.05) there was intense staining for {alpha}6 in the basement membrane of luminal epithelium and the shallow glands (Figs. 3, A and B, and 4, A and B). Although the median staining intensity scores for the basement membrane of luminal and glandular epithelia were the same for metestrous and diestrous samples, overall scores were significantly different (P < 0.05), because diestrous samples consistently scored lower. Expression declined in these regions in proestrous and estrous samples (P < 0.05; Fig. 3, C and D, and Fig. 4, A and B). Diffuse staining of the luminal epithelium for {alpha}6 declined at proestrus and metestrus (P < 0.05; Fig. 4C). No difference in staining pattern was observed between OVX-treated and untreated animals (P > 0.05; Fig. 3E).



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  		FIG. 3. Immunohistochemical localization of the integrin subunit {alpha}6 in cryosections of bovine endometrium (A–E). Immunofluorescent co-localization of {alpha}6 (G-Alexa 488 tag) and laminin (H-Alexa 594 tag) on a section prepared from a Day 15 animal. Immunostaining (indicated by black deposit) was detected in stromal cells (S) and luminal (LE) and glandular (GE) epithelia and vasculature. {alpha}6 antibody also reacted with lateral and apical surfaces of epithelial cells. A) Day 3, B) Day 17, C) Day 18, D) Day 19, E) OVX-E+P, F) negative control treated with rat IgG. Arrow indicates capillary endothelial cell staining. Nuclei counterstained with hematoxylin. The bars represent 50 µm



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  		FIG. 4. Estrous cycle effect on {alpha}6 expression in luminal epithelium (LE) basement membrane (A), shallow glands (SG; B), and luminal epithelium cells (C). Bars with different letters are significantly different (P < 0.05) from other stages of the cycle

Cycle-Independent Integrin Expression

Moderate expression of the {alpha}3 subunit was detected throughout the cycle on endometrial epithelium (Fig. 5A). Staining was confined to epithelial cell surfaces, and expression did not appear to be affected by the ovarian steroids. Staining patterns were similar in all OVX animals (Fig. 5B). Integrin subunit {alpha}4 was localized to stromal and endothelial cells in cycling (Fig. 5C) and ovariectomized animals (Fig. 5D). Scattered staining was present throughout the stroma, and concentrated staining occurred in lymphocytic foci in some sections (Fig. 5D).



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  		FIG. 5. Immunohistochemical localization of cycle-independent integrins {alpha}3 (A and B) and {alpha}4 (C and D). Staining for {alpha}3 was present on luminal (LE) and glandular epithelia (GE) in cycling (A) and OVX (B) animals. Staining for integrin {alpha}4 was present on subepithelial stromal cells (S) in cycling and OVX animals (D). Arrow, lymphocytic focus. Brown deposit indicates antibody reactivity; nuclei counterstained with hematoxylin. The bars represent 50 µm.

FIG. 6. Immunohistochemical localization of collagen IV and fibronectin in cryosections of bovine endometrium. Immunostaining for collagen IV (A) was present in the subepithelial stroma (S) but not the basement membrane of luminal epithelium (LE). It was observed in the basement membranes of glandular epithelium (GE) and vasculature (not shown). Staining for fibronectin (B) was present in the subepithelial stroma. Decreased reactivity was detected in deeper caruncular (CAR) stroma. Bars represent 50 µm. ICAR, intercaruncular region.

The distribution of ß1 overlapped staining patterns observed for {alpha}3, {alpha}4 and {alpha}6. Immunoreactivity occurred in the basement membrane of glandular epithelium and endothelium, resembling {alpha}6 staining patterns. Anti-ß1 also stained luminal epithelium and subepithelial stromal cells, consistent with {alpha}3 and {alpha}4 distributions, respectively. No differences in ß1 expression or distribution were observed between the cycle days examined, nor did these patterns differ for OVX animals.

ECM Distribution in Bovine Endometrium

Immunofluorescent co-localization of {alpha}6 and laminin revealed parallel patterns of distribution and expression, but pericellular staining of luminal epithelium was detected only in sections treated with anti-{alpha}6 (Fig. 3G). Laminin was detected in the basement membrane of epithelium and blood vessels (Fig. 3H). Collagen IV antibodies reacted with the basement membranes of endothelium and glandular epithelium. Reactivity was also present on subepithelial stromal cells and the basement membrane region of luminal epithelium (Fig. 6A). As shown in Figure 6B, fibronectin was present in the stroma and blood vessels, but not in uterine epithelium, and expression was minimal in caruncular endometrium. Distribution and expression of collagen IV and fibronectin did not vary during the estrous cycle. Distribution patterns for integrins and ECM proteins in bovine endometrium are summarized in Table 2.


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  		TABLE 2. Integrin and ECM distribution in bovine endometrium.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the luminal epithelium was found to express integrin subunits ß1, {alpha}3, {alpha}6, and integrin {alpha}vß3. Of these integrins, {alpha}6 and {alpha}vß3 exhibited estrous cycle-dependent expression and distribution. The {alpha}6 integrin subunit associates with either ß1 or ß4 subunits [23]. The distribution of {alpha}6 and ß1 subunits in the basement membrane of glandular epithelium and blood vessels suggests that {alpha}6ß1, a laminin receptor, is present here. This distribution correlates with the immunohistochemical localization of laminin to the basal lamina of epithelium and vasculature. Localization of ß4 was not undertaken in this study because of limited cross-reactivity of available antibodies with bovine ß4. However, ß4 is known to heterodimerize with {alpha}6 in the basement membrane of epithelium to form the {alpha}6ß4 laminin receptor that is central to hemidesmosome formation [24, 25]. Thus it is probable that both {alpha}6ß1 and {alpha}6ß4 heterodimers are present in uterine epithelium. Estrogen may decrease {alpha}6 expression. The temporal regulation of {alpha}6 in the basement membrane of uterine epithelium coincides with increasing estrogen levels and, as a result, expression of P4 and estrogen receptors [26]. In sheep, estrogen and P4 receptors first appear on the luminal epithelium and then develop on the glandular epithelium [27]. If similar estrogen and P4 receptor development occurs in cattle, these transcription factors could be either directly or indirectly involved in the down-regulation of {alpha}6. In humans, {alpha}6 undergoes a cycle-specific change in distribution, appearing on the lateral membranes of luminal epithelium during the secretory stage, which corresponds to the diestrous stage in cattle [28].

This study revealed a unique spatial and temporal pattern of {alpha}vß3 expression in cows in comparison to humans and pigs. Integrin {alpha}vß3 is considered to be a marker of uterine receptivity in women, since it appears five days post-ovulation on luminal epithelium for a period corresponding to the implantation window [29]. Its delayed appearance in women has been correlated with infertility [29]. In pigs, expression of {alpha}v and ß3 subunits was localized to luminal epithelium; strong expression of ß3 occurred at the apical surface, and {alpha}v was present on epithelial cell borders [18]. In the present study, staining for {alpha}vß3 in bovine endometrium was strongest in the luminal epithelium basement membrane region and underlying subepithelial stromal cells at proestrus, estrus, and metestrus. During diestrus, subepithelial stromal staining was reduced, and on Day 16 little or no expression was detected in these regions. In OVX animals, both estrogen and P4 were associated with reduced expression of {alpha}vß3.

In vitro experiments have demonstrated a direct relationship between sex steroids and {alpha}vß3 expression in human cells. Cultured endometrial adenocarcinoma cells (Ishikawa cells), exposed to estrogen or pretreated with E2 followed by P4 exposure, down-regulated {alpha}vß3 [30, 31]. Conversely, Sillem et al. [32] were unable to demonstrate steroid regulation of integrin receptors using primary endometrial cell cultures. However, the cells in these cultures were not polarized, and previous workers [33] demonstrated that nonpolarized uterine epithelium is unable to respond to steroid hormones. Although the studies with Ishikawa cells are consistent with the results in OVX animals, the cyclic and regional differences in {alpha}vß3 expression in bovine endometrium suggest a more local form of regulation. In sheep, cell- and tissue-specific expression of estrogen and P4 receptors has been demonstrated [27], so that the steroids may be influencing cell phenotype. However, it is well established in other cell systems that a number of growth factors influence {alpha}vß3 expression [31, 34–36], and that the growth factors themselves are regulated by the sex steroids [37]. Expression of transforming growth factor (TGF)-ß, interferon-{tau}, basic fibroblast growth factor, and TGF-{alpha} varies according to cell type and cycle stage in caprine endometrium [38]. Thus it is probably a combination of the ovarian steroids and growth factors that regulates integrin expression.

Reduction of integrin {alpha}vß3 on Day 16, the day of pregnancy recognition, coincides with a change in the hormonal environment in cycling animals, including increased oxytocin receptor development and activation of the prostaglandin synthetic pathway [39–41]. It is possible that prostaglandin release in response to oxytocin may directly influence {alpha}vß3 expression on Day 16. In other cell systems, prostaglandin E2 causes decreased transcription of the ß3 subunit [42], the limiting factor in {alpha}vß3 receptor assembly. The abrupt change in the prostaglandin E2 and F2{alpha} ratio could have a negative impact on {alpha}vß3 expression, causing down-regulation on Day 16. Furthermore, Asselin et al. [43] have demonstrated differential expression of prostaglandin E2 and F2{alpha} in cultured bovine caruncular and intercaruncular endometrium. Perhaps regional differences in prostaglandin levels are important in the differential expression of {alpha}vß3 in caruncular and intercaruncular endometrium. Of interest is the restriction of expression of {alpha}vß3 to intercaruncular endometrium, suggesting that it may act to prevent villus development.

A downstream event that occurs in response to prostaglandin release in human ciliary muscle cells is prostaglandin-stimulated matrix metalloproteinase activity [44, 45], which affects ECM composition and is enhanced by low levels of estrogen [46]. Conceivably similar processes could occur in response to prostaglandins in bovine endometrium.

Constitutively expressed integrin subunits included {alpha}3, {alpha}4, and ß1. Subunits {alpha}3 and {alpha}4 heterodimerize exclusively with ß1, forming collagen/laminin/{alpha}2ß1 and fibronectin/VCAM-1 receptors, respectively [47]. As demonstrated by immunohistochemical studies in endometrium obtained from cycling baboons, humans, pigs, and now in cattle, {alpha}3ß1 expression is not affected by the ovarian steroids. This observation is validated by constant expression in ovariectomized gilts [18], baboons [48], and, in the present study, cows. The cytoplasmic epithelial staining observed in all these species is consistent also with {alpha}3ß1 distribution observed in other tissue types [7]. The across-cell distribution of {alpha}3 suggests that on lateral borders it may be involved in cell-cell adhesion to {alpha}2ß1, as has been observed in keratinocytes [49, 50]. There may also be binding of {alpha}3ß1 to laminin in the basement membrane of epithelial cells.

Contrary to the modulated epithelial distribution of {alpha}4 demonstrated in humans, baboons, and pigs [19, 48, 18], {alpha}4 was not observed in bovine uterine epithelium. It was expressed exclusively in bovine stroma, and expression was constant throughout the cycle and in treated and untreated ovariectomized heifers. Reactive cells in the stroma may be of immune cell lineage, since some lymphocytes express {alpha}4ß1 [7]. Integrin subunit {alpha}4 was an excellent marker of lymphoid aggregates in bovine endometrium: these foci have been reported in healthy endometrium previously [51]. Overall, the constitutive expression of {alpha}4 indicates that it is not a key integrin for the cellular interactions that lead to embryo attachment.

The integrin subunit ß1 is able to heterodimerize with 12 {alpha} subunits so that one class of {alpha}ß1 may be down-regulated while another is up-regulated. Its distribution within bovine endometrium overlapped with cells expressing {alpha}3, {alpha}4, and {alpha}6, indicating that it probably forms receptors with these subunits. The ECM proteins examined in this study included laminin, collagen type IV, and fibronectin. As was anticipated, laminin was localized to the basement membrane of epithelium and vasculature. Collagen IV antibodies reacted with subepithelial stromal cells, and the basement membrane of glands and blood vessels. Similar distributions have been observed in human endometrial samples, with collagen IV being detected in stromal cells and the basement membrane of glands and blood vessels [52, 53]. Localization of collagen IV to the basement membrane region of luminal epithelium is a distribution pattern previously only observed in baboon endometrium [48]. Fibronectin was found to be distributed diffusely throughout the loose connective tissue stroma, but lower levels were detected in deeper caruncular endometrium. Like fibronectin, {alpha}vß3 was not present in caruncles, suggesting that {alpha}vß3 may bind fibronectin in intercaruncular stroma. Other ligands not examined that may have been informative about {alpha}vß3 interactions include vitronectin and osteopontin. Osteopontin exhibits distribution patterns parallel to those of {alpha}vß3 in baboon endometrium [48], and vitronectin was co-expressed with {alpha}vß3 at the apical surface of porcine epithelium [18].

Endometrial integrin expression is clearly species-specific, which probably reflects differences in estrous cycles and placentation among the animals studied to date. Unlike the situation in humans, bovine endometrium does not undergo cyclic shedding and repair, nor is the implantation process invasive. Binucleate cell fusion with maternal uterine epithelium is the limit to trophoblast invasion [54]. In view of these fundamental differences, it is remarkable that integrins {alpha}6ß1 and {alpha}vß3 are modulated in both the bovine estrous cycle and the human menstrual cycle. Whether this is coincidental or shows that these receptors are sensitive to regulation by the sex steroids remains to be elucidated.

In conclusion, this study has shown that integrin expression is modulated during the bovine estrous cycle and that there is a repertoire of constantly expressed integrins and ECM proteins. Although it is not known what factors contribute to integrin expression, potential modulators include ovarian steroids, ECM, growth factors, and prostaglandins.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Hai-Choo Lim for her expert technical assistance and Dr. Tess Astatkie for his help with statistical analysis. We are also grateful for the PRIDs provided by Drs. Greg Keefe and Rob Lofstedt, Atlantic Veterinary College, and Lutalyse generously donated by Pharmacia-Upjohn, Mississauga, ON, Canada.


   FOOTNOTES
 
1 This work was supported by the Natural Sciences and Engineering Research Council, Nova Scotia Department of Agriculture and Marketing, Dairy Farmers of Canada. Back

2 Correspondence: Leslie A. MacLaren, Department of Animal Science, Nova Scotia Agricultural College, Box 550, Truro, NS, Canada B2N 5B1. FAX: 902 895 6734; l.maclaren{at}nsac.ns.ca Back

Accepted: June 17, 1999.

Received: May 5, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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